TWI345472B - Methods of diagnosis of spinal muscular atrophy and treatments thereof - Google Patents

Description

1345472 IX. Description of the Invention: [Technical Field] The present invention relates to a method for treating a neurological disease, particularly a spinal muscular atrophy (SMA). [Prior Art] Survival Motor Neuron Protein

SMN) is generally considered to be a protein involved in axonal processing (ax〇nai pr〇cessing). Motor neurons lacking SMN have small growth cones and defects in axonal outgrowth. However, until today, we have almost no knowledge of this defect and its role in the pathogenesis of spinal muscular atrophy. 0 Spinal muscular atrophy is a somatic recessive genetic disease characterized by ridges. The motor neurons of Zhao Qianjiao degenerate, causing muscle atrophy and paralysis. It has been confirmed that the gene causing proximal spinal muscular atrophy is a homozygous or a deletion mutation of the SMN1 (survival motor neuron 1) gene (Lefebvre et. al. (1995) Ce// 80: 155-165 ), because it is closer to telomeres, it is also called a telomeric copy of the SMN gene. The SMN2 (survival motor neuron 2) gene, also known as the centromeric copy of the SMN gene, is almost identical to SMN1, however both can be distinguished from the nucleotide differences between exons 7 and 8. Since SMN1 produces more than 90% of full-length mRNA, and most of the SMN2 transcript lacks exon 7, and it encodes a C-terminal truncated SMN protein, SMN2 is not functionally capable of replacing SMN1 (Monaniet. al. ( 1999) Hum Mol Genet%: 1177-1183; Lorson et. al. (1999) Proc Natl 1345472 «Sc/ 96: 6307-6311). This SMN protein line is widely expressed in all tissues and is particularly highly expressed in spinal motor neurons. The SMN line is related to the severity of the disease (Lefebvre et. al. ΛΜ 265-9 (1997); Coovert et. al. (1997) Hum Mol Genet 6* 1205-14) and is already small in spinal muscular atrophy Proved in the mouse model (Hsieh-Li et al. (2000) JWzi 24, 66-70; and Monani et. al (2000) Human Mol Genet 9, 2451-7) °

Recent studies indicate that SMN may play a key role in the biosynthesis of spliceosomal small nuclear ribonucleoprotein (snRNP) in all cell types (Liu et. al. (1996) Embo J 15, 3555- 65; Pellizzoni et. al. (1998) Cell 95, 615-24; and Pellizzoni et. al. (2002) iScz'ewce 298, 1775_9). However, the mechanism of action of SMN protein deficiency leading to motor neuron degeneration remains to be elucidated. In recent years, there have been numerous journal articles dedicated to the study of the neuron-specific functions of SMN. It was found that motor neuron lines lacking the SMN protein showed shorter axons, smaller growth cones, and the transmission of axonal mRNA and neuromuscular formation were defective. This phenomenon indicates that SMN proteins play an important role in neuronal cells (Fan et. al. (2002) //wm Mo/ Gewei 11, 1605-14; Rossoll et. al. (2003) J Cell Biol 163, 801-12 i Zhang et al. (2003) iVewrosci 23, 6627-37; and McWhorter et. al. (2003) J Ce//price/162, 919-31). Axonal transport obstruction in motor neurons may result in inability to perform protein translation and axonal growth. This phenomenon suggests that certain mechanisms, such as microtubule assemblies, are important in axonal maintenance. 1345472 Microtubules are important for the formation of neuronal cytoskeleton. Its role is in the regulation of neuronal transport, the maintenance of neurites, and the regulation of ax〇nal elongation and growth cone steering. Two types of proteins are involved in the regulation of microtubule dynamics, one of which stabilizes microtubules and the other that disrupts microtubule stability. Structural microtubule associated proteins (MAPs) are via microtubules

The combination of polymer lattices, their interaction with microtubules, and the interaction of microtubules with actin to promote microtubule assembly (Hir〇kawa, Ν·(1994) Cw" 〇/ / Price / 6, 74-81, and Matus, A. (1988) 29-4). Recently, proteins that are involved in disrupting microtubule stability such as cytosolic protein and SCG10 have also been discovered (Sobel, A. (1991) Biochem Sci 16, 301-5; Belmont et. al. (1996) Cell 84, 623-31; And Riederer et al. (1997) ¢/5/4 94, 741-5). Cytoplasmic protein/〇 pl8 is phase-specific in most cell types and is highly expressed in the developing nervous system (Riederer et. al. (1997) Proc Natl Acad Sci USA 94, 741-5; Schubart , UK (1988) J Biol Chem 263, 12156-60; Koppel et. al. (1990) JC/zem 265, 3703-7; and Amat et. al. (1991) Price Z) ev 60, 205-18) And will be down-regulated in terminal differentiation (Amat et. al. (1991) 60, 205-18 ! Schubar et. al. (1992) Differentiation 51, 21-32; Amat et. al. (1990) Mo/ and eproi/ Dev 26, 383-90). Cytosolic protein promotes microtubule depolymerization by increasing the rate of microtubule decomposition (Belmont et. al. (1996) Cell 84, 623-31; Amat et. al. (1991) Brain Res Dev Brain Res 1345472 60, 205-18). The cytosolic protein system binds to the tubulin dimer to form a ternary complex. This reaction may result in inhibition of microtubule assembly by reducing the free state of tubulin concentration, resulting in a decrease in microtubule elongation (Belmont , LD & Mitchison, TJ (1996) Cell 84, 623-31) 〇 [Summary of the Invention]

The present invention provides a method of treating spinal muscular atrophy in a patient comprising administering to the aforementioned patient a genetic recombination vector comprising at least one copy of a cytosolic prion inhibitor. The suppressor can be a cytosolic protein expression repressor. In some embodiments, the expression inhibitor is an antisense nucleic acid sequence or an RNAi nucleic acid (e.g., siRNA or shRNA), or may be encoded with the aforementioned antisense nucleic acid sequence or RNAi nucleic acid. The copy may be a copy of a cytoplasmic protein inhibitor which is manifestable in the host' or a cytosolic protein expression inhibitor which is manifested in the host.

In some embodiments, a 'nucleic acid vector is used herein to increase or decrease the amount of transcription/expression of cytosolic proteins, and/or to introduce nucleic acids, wherein the nucleic acid is reinforced with a cell A gene encoding a prion protein (such as a constitutively active cytoplasmic protein), or a gene encoding a cytoplasmic protein expression in a knock-down target cell. The present invention provides a method for diagnosing a condition caused by spinal muscular atrophy or a low motor neuron survival protein (l〇w SMN protein-based condition). In some embodiments, a method comprising the steps of: providing a cytosolic protein binding agent and a cell sample obtained from a human patient, wherein the aforementioned human patient is at least suspected of having spinal cord 1345472 muscular muscular dystrophy Or a condition caused by a low motor neuron survivin; contacting the aforementioned cytoplasmic protein binding agent with the aforementioned cell sample under conditions in which the binding agent can specifically bind to the cytoplasmic protein in the sample to produce a complex' And removing the binding agent that does not produce specific binding to leave the remaining complex, and detecting the remaining complex, and comparing the detected amount of the complex in the aforementioned sample or the corresponding cytosolic prion protein concentration in the healthy cell sample Control amount. The increase in the amount of complex or the increase in cytosolic prion protein was detected as a positive screening result. In some embodiments, the cytosolic protein binding agent can be

An anti-cytoplasmic protein antibody or a cytoplasmic protein binding agent that has been labeled with a detectable label. In some embodiments, the screening results are combined with further test results to diagnose conditions caused by spinal muscular atrophy or low motor neuron survival proteins. The further test result includes a test result of the performance of the functional low motor neuron survivin, a nuclear "gem", a test result of the occurrence of a nuclear gem, a test result of the amount of snRNp formation, or The present invention provides a kit for use in any of the diagnostic or screening methods described herein. In some embodiments, a wipe is provided for the sample (10). The presence of a protein (eg, high expression, normal nail, low, or no cytoplasmic prion protein. The application of the present invention will be understood from the description provided herein. The description of the technical features of the present invention is only for the purpose of illustrating the nature, manufacture and use of the subject matter of one or more aspects of the present invention, and is not intended to limit the scope of the present invention. The present invention is directed to the scope of the application, the particular application, or the application of the priority of the present application, or the scope, application, or use of the invention. The following definitions and non-limiting criteria are used to examine the present invention. The description is necessary. In particular, although the discussion of some aspects of the present invention is related to the treatment of spinal muscular atrophy, these discussions are not intended to limit the invention to 'beta-beta'. The headings (such as "previous technology" and "invention content") and subtitles are only used in this specification as a general organization topic, and are not intended to limit the technology of i invention or any of its viewpoints. In particular, in "previous technology" The subject matter of the specification may be included in the present specification. The material classification or discussion of the material has a specific utility for ease of explanation. It cannot be inferred from the classification of the material herein that the material must have this function or only The references cited in the present disclosure are not admitted to be prior art or related to the patentability of the technology of the present invention. The documents cited in the "Embodiment" section of the specification are for various purposes, and are incorporated herein by reference. If the texts and similar materials contained in one of the references or XOR, including but not limited to the definition of terms, the use of terms, the techniques of description, etc., are subject to the disclosure of this application.

The description of the embodiments and the specific embodiments are intended to be illustrative only and not to limit the scope of the present invention. In addition, the plurals mentioned in the text have the characteristics described below, and are not intended to exclude other implementations with additional features. The disclosure provides a composition and method for how to make and use the techniques of the present invention, which are not indicated by the embodiments of the present invention, which are not produced or tested, or otherwise produced or tested. Part of the implementation. Items Unless otherwise stated, all percentages of combinations referred to in this specification are based on the weight of the total composition. In this document, the words "including," and their variants are non-limiting, as the items listed in the list do not exclude other substances, compositions, or methods that may be used in the techniques of the present invention.

The present invention provides a method for treating spinal muscular atrophy in mammals, a method for diagnosing spinal muscular atrophy in mammals, and a kit for screening spinal muscular atrophy in mammals. In some embodiments, the mammal is a human. In some embodiments, a method of treating spinal muscular atrophy comprises administering a genetic recombination vector comprising at least one copy of a cytoplasmic protein inhibitor. The cytosolic protein is a highly-conserved protein having a molecular weight of about 17 kDa (see SEQ ID N 0.3). Its function as an important regulatory protein for microtubule dynamics has been explored in detail. Microtubules have a high dynamic structure and are continuously assembled and disassembled continuously. Cytoplasmic proteins play an important role in rapid microtubule reorganization in response to the cytoskeleton required by cells. The microtubule is a cylindrical polymer composed of α, 1345472 - tubulin, and the microtubule assembly is partly determined by the concentration of free tubulin in the cytoplasm. When the free tubulin concentration is low, the growth rate at the end of the microtubules is slowed, resulting in an increase in the rate of depolymerization, that is, an increase in disassembly.

The cytosolic protein interacts with two alpha, no-tubulin binary molecules to form a tight ternary complex called the T2S complex. A cytoplasmic cytoplasmic protein and a bimolal tubulin binary system are bound by a cytoplasmic-like domain (SLD), and when cytosolic globin is combined with tubulin In the T2S complex, tubulin becomes unpolymerizable, and without the polymerization of tubulin, there is no microtubule assembly. Through this mechanism, cytosolic prion protein promotes de-assembly of microtubules without directly acting on the ends of microtubules. Microtubule assembly rate is important in cell growth, so regulation of cytoplasmic prion protein is also involved in cell cycle progression. The regulation of cytoplasmic proteins depends on the cell cycle and is controlled by cellular protein kinases that respond to specific cellular signals. Phosphorylation of the four serine residues Serl6, Ser25, Ser38, and Ser63 of cytosolic proteins leads to weakening of the binding of cytosolic protein-tubulin, and the acidification of cytosolic proteins is increased in the cytoplasm. The concentration of tubulin that can be used for microtubule assembly. For the initiation of the mitotic phase of the cell cycle, a mitotic spindle is necessary, and for the cells to be assembled with the cell division spindle, phosphorylation of cytosolic prion must occur. Without the growth or assembly of microtubules, the cell division spindle cannot form and the cell cycle ceases. In the cytokinesis of the last period of the cell cycle, cytosolic prion protein is rapidly de-acidified, preventing cells from entering the cell cycle before the time of maturity 1345472.

High or excessive expression of cytosolic prion protein in a cell according to the present invention results in the death of motor neurons in the cell. In addition, increased performance, high performance, or overexpression of cytoplasmic prion is a sign of spinal muscular atrophy. Therefore, the amount of cytosolic protein expression can be used as a biomarker for screening for spinal myelodysplastic disease. Furthermore, knockdown SMN performance is associated with up-regulation of cytoplasmic protein levels. Up-regulation of the amount of cytosolic prion protein in 'SMA-like mice' in animal models disrupts microtubule maintenance during axonal growth, leading to axonal degeneration (axon degeneration), Axonal lesions are manifested in the diaphragm muscle of mice with spinal muscular atrophy. In addition, increased cytosolic prion protein in the spinal cord muscular atrophy mice in the control group was accumulated in the ridge motor neurons. According to the present invention, the use of proteosome analysis revealed that cytoplasmic prion protein, which disrupts the microtubule stabilizing factor, is up-regulated in SMN knockdown NSC34 cells. Excessive expression of cytosolic proteins disrupts the organization of microtubules in NSC34 cells. Furthermore, in motoneurons cultured in primary culture, excessive expression of cytosolic prion protein can cause dying-back axonal damage, and highly expressed cytoplasmic prion protein will stretch in axons (ax〇n Cause damage in the extension). In addition, accumulation of cytoplasmic proteins in motor neurons results in high golgi fragmentation and death of motor neurons. Therefore, the excessive expression of 'cytoplasmic prion protein can cause internal and external damage to motor neurons'. It is thus known that cytoplasmic prion protein is involved in the pathogenesis of spinal muscular atrophy. Furthermore, the damage in the axon extension of motor neurons is related to the destruction of microtubules. In some embodiments, reference is made to a method of treating spinal muscular atrophy in a patient comprising administering to the patient a genetically recombinant vector comprising at least one copy of a cytosolic prion inhibitor. The suppressor can be a cytosolic protein expression repressor. In some embodiments, the expression inhibitor is an antisense nucleic acid sequence or an RNAi nucleic acid (e.g., siRNA or shRNA), or may be encoded with the aforementioned antisense nucleic acid sequence or RNAi nucleic acid. The copy may be a copy of a cytoplasmic protein inhibitor which is manifestable in the host' or a cytosolic protein expression inhibitor which is manifested in the host. In some embodiments, the target sequence of the antisense nucleic acid sequence or RNAi nucleic acid is CGT TTG CGA GAG AAG GAT A (SEQ ID. N0.6).

In some embodiments, the nucleic acid vector can be used herein to increase or decrease the amount of transcription/expression of cytosolic proteins and/or to introduce nucleic acids, wherein the nucleic acid is reinforced with a cytoplasmic protein (eg, having a constant activity) The gene encoding the cytoplasmic protein, or a gene encoding a cytoplasmic protein in the target cell. "Knockdown" refers to the introduction of a nucleobase polymer (such as a nucleic acid) that reduces the amount of expression of a selected target gene into a cell; this is related to gene knock-out and gene maturation ( Gene silencing) differs in the way that the target gene is completely excluded. The knockdown of cytosolic prion protein can be performed using RNAi technology, such as introducing the following items into cells: (1) siRNA or morpholino oligomeric substance targeting cytosolic protein RNA, the amount of which is controlled, or (2) A construct expressed in a host that encodes a gene encoding a shRNA that is a cytoplasmic protein RNA. In some embodiments, the shRNA derived from the target cytosolic protein RNA can be expressed by a nucleic acid, and can also be used for this purpose. For example, MISSION shRNA 1345472 f

Nucleic acids (RNA1 nucleic acids from Yijj, available from sigma_Aidrich, Inc., St Louis, MO, USA) can also be used according to the manufacturer's instructions. This exemplified sequence with the shRNA gene encoding is attached to a promoter (such as the U6 promoter). The structure to be constructed is sent to the cells for transport into the nucleus and is expressed. The sequence of the RNAi nucleic acid used to prepare the knockdown cytoplasmic protein can be readily obtained, for example, from SEQ ID Nos: 1 and 2, and can be prepared by methods known in the art 'as in Ui-Tei et. al. (20〇4) M/c/· Xc Secret 32: 936-48. The 'identified shRNA sequences can then be introduced into the construct and sent (e. g., via a vector) to motor neuron cells. Such a method for knockdown cytosolic protein is as described in EpStein_Barr et. al. (2006) Μ?/. Ce//. 17(7): 2921-2930.

The Epstein-Barr viral vector obtains a constant expression of shRNA targeting the cytoplasmic protein. A shRNA nucleic acid or a sequence thereof which is commercially available as a cytoplasmic protein can also be used. For example, SureSilencing shRNA STMN1 LAP18/Lag Human Stathmin 1/oncoprotein 18 (shRNA targeting cytosolic protein, available from SuperArray Bioscience Corporation, Frederick, MD, USA), and HuSH 29mer shRNA construct against STMN1 (production) Serial No. TR318815, available from OriGene Technologies, Inc., Rockville, MS, USA). The manner in which the cytoplasmic prion protein expression described herein can be replaced by the remaining cytoplasmic prion protein expression can be used. For example, in some embodiments, an antisense nucleic acid sequence of cytoplasmic protein or an siRNA-based cytosin gene miniaturization strategy can also be used. See, for example, Alii et. al. (Aug 1345472 14, 2006) [Internet publication prior to printing]. Alternatively, cytosolic prion RNA decomposing activity (e. g., anti-plasmon ribozyme) can be expressed by introducing a recombinant construct into a target cell. See, for example, Mistry et. ai. (2001) Antisense Nucl. Drug Dev. 11(1): 41-49 °

A vector can be used to deliver a nucleic acid construct into a motor neuron cell. In some embodiments, the vector may be a recombinant viral vector comprising all or part of a complementary sequence of a viral chromosomal nucleic acid. See, for example, Federid et. (2006) Mwsc/e «δ 33(3): 302 (2006). In some embodiments of the invention, if a lethal virus is used as or forms a viral vector, these vectors contain a partial complement of the viral chromosome and may be non-lethal. Viruses that can be used to form recombinant viral vectors include Adenovirus (AV), Adeno-Associated Viruses (AAV), herpes viruses, and ientiviral vectors, which can be non-lethal. of. See, for example, Haase et. al. (1997) Nature Med. 3: 429-436; Vincent et. al. (2004) Neuromolec. Med. 6(2-3): 79-85; Latchman et. al. (1999) JR A/ei/· 92(11): 566-570; and Wong et. al. (2006) 77zer. 17(1): 1-9. In some embodiments, the virus exemplified as the preparation of the viral vector may be: a first or second generation adenovirus or an Epstein-Bar virus or a herpes simplex virus. In some embodiments, AAV or a second generation AV can be used to form a recombinant viral vector herein. See, for example, Barton et. al. (2006) Mo/ec. 77zer 13: 347-56; and Alba et. al. (2005) 12 Suppl. (1): S18-27 (Oct 2005). Such a gene vector can be administered one or more times during a course of treatment, and each time 1345472 can be administered the same vector or a different vector. Such a carrier may be combined with a non-primary carrier therapeutic agent, the therapeutic agent package; in a manner of being fun, nutritionally, or otherwise medically acceptable beneficial substance, 'this further treatment The agent may be one or more or indirectly inhibiting the transcription of the cytoplasmic protein gene, the treatment of the cytoplasmic prion protein/primary, the expression of the cytoplasmic prion protein, or any of the cytoplasmic substances. Activity of the cytoplasmic protein

A method for diagnosing vertebral muscular atrophy or a low-handed sample includes the following steps: providing a cytoplasmic protein:: J and a sample of cells obtained from a human patient, wherein the two are atrophy Or low motor neurons; egg: prion protein binding agent and the aforementioned cell sample in the ί: 峨 protein specific contact ^ ^ contact contact shouting complex, and removed unproduced

Residual!: Complex; and detection of residual complex 2 or corresponding cytoplasmic protein concentration and health: Control 1 in the cell sample. Among them, the test results of the composite test. In part of the implementation, the above-mentioned test = anti-thinking or additional testable screening results are further improved; in the trial implementation, 'the aforementioned meat atrophy or low motor neuron deposits', two of the 2 broken muscle steps The test, the gentleman's fruit in the ^ a, raised the disease. The further test results, the nuclear m low motoneuron survival protein performance in the nuclear gem black, the appearance of the test results, 1345472 snRNP formation The test result of the quantity, or a combination thereof.

The present invention provides a kit for use in any of the diagnostic or screening methods described herein. In some embodiments, a method is provided for determining the presence of cytoplasmic prion protein in a test sample (eg, high performance, normal, low, or absence of cytoplasmic prion protein. The sample source may be a test subject or A patient who may have spinal muscular atrophy. In some embodiments, the sample may be an embryo to determine the risk of spinal muscular atrophy after birth in a mammal. A component comprising two or more diagnostic assays. In some embodiments, the component can be a compound, reagent, container, and/or implement. For example, a container in a set May contain an antibody or fragment thereof that specifically binds to cytoplasmic proteins. Such antibodies or fragments may be provided by attachment to a support material. One or more additional containers may be attached for the assay. An element such as a reagent or a buffer. In some embodiments, the kit may contain a detection reagent, wherein the detection reagent has a reporter group or a straight line. Or indirectly detecting the labeling of antibody binding. In some embodiments, the kit may include instructions for screening for an increase in the concentration of cytosolic prion protein. The kit may include combining one screening result with at least one more Further testing or screening instructions to diagnose a condition caused by spinal muscular atrophy or low motor neuron survival protein. In some embodiments, the further test or screening result includes functional SMN protein expression Test results, test results of the presence of "gem" dot structures in the nucleus, test results of the amount of snRNP formation, or a combination thereof. The kit can be used to screen the spinal cord on a mammalian test subject (such as a human) 19 1345472 Muscle Atrophy. EMBODIMENT The present invention is further understood by the following examples, which are not intended to limit the scope of the invention in any way.

Construct: The SMN shRNA system was constructed by two chemically synthesized oligonucleotides, GATCCCGTAGCTAATAGTACAGATCCAAGAGAGTTCT GTACTATTAGCTACTTTTTT (SEQ ID N0.4) and 'AGCTTAAAAAAGTAGCTAATAGTACAGAACTCTCTT GAAGTTCTGTACTATTAGCCAT (SEQ IDN0.5), bonded It is loaded into the Bglll position of the pSuperior.puro carrier (OligoEngine) and the Hindlll position, which is then referred to as pSuperior-SMN shRNA. Expression of the EGFP-epitope epithelial pEGFP-cytoplasmic prion protein The full-length cytoplasmic prion protein cDNA (SEQ ID. N0.1) was cloned into pEGFP in the correct reading frame (in frame). BamHI position of the C3 vector (BD Biosciences). The 45 kb cytoplasmic protein coding region fragment was cloned into the EcoRI position of the pCMV2-Flag vector (Sigma). Selection of cultured cells, transfection, and stable selection: NSC34 cell lines were cultured daily in DMEM medium (Dulbecco, s modified Eagle's medium), and the DMEM medium was added with 10% heating. Heat-inactivated fetal calf serum (Fetal Bovine Serum, FBS), lmM glutamine, and 100 IU/ml (S) 20 1345472

Penicillin was cultured with 100# g/ml Key Technologies (Life Technologies) and the cell line was humidified at 5% C〇2 in a humidified atmosphere at 37 °C. Transfection was performed via the use of Lip〇fectAMINETM2000 (Invitrogen) according to the manufacturer's recommendations. To produce a stable selection of inducible RNAi, NSC34 cells were first transfected with the pcDNA6/TR vector (Invitrogen) and an independent anti-Blasticidin strain was selected. Next, the NSC34-TR cell line was transfected with pSuperior-SMN shRNA, and a single selection strain was selected by adding 1 " g/mi puromycin. Finally, the amount of SMN in stable colonies treated with or without viaoxycycline was determined by Western blot. The isolation and primary culture of motoneurons was performed according to the description of Wiese et. al. (1999) 513, 1668-76; and Duong et. al. (1999) Price «/ corpse krwflco/128, 1385-92. Primary motor neuron

The transfection lines were carried out according to the procedure described by Scherer et. al. (2002) Med 4, 634-43 using 0.1 M polyethylenimine reagent (PEI, Sigma). Briefly, plastid DNA diluted by 〇pti_MEM (Gibco) (cytosolic serotonin-eGFp or eGFP) is added to the same amount of polyethyleneimine solution diluted in 〇pti_MEM (〇.1M). PEI' is dissolved in 5% glucose) (the N/P ratio is 10). After 15 minutes of incubation, the above mixture was added to the culture solution. After 36 hours of transfection, the cells were harvested for immunocytochemical analysis. Proteomic analysis: spinal cord from spinal cord muscular atrophy mice with SMN knockdown NSC34 cells or control mice with control cells in lysis buffer (l50 mM NaC 50 mM Tris/HCl pH 8.0, 2 mM) EDTA, 0.5% sodium deoxycholate, 1% Nonidet 1345472 0

Cell lysis was carried out by hydrazine, 1 mM NasVO4, 50 mM NaF), wherein the aforementioned lysis buffer was supplemented with a mixed protease inhibitor (Rhoche). Then, the cell lysate was centrifuged at 12,000 rpm for 20 minutes using a cold-keeping centrifuge, and the supernatant was collected and precipitated by adding 10% trichloroacetic acid (TCA) dissolved in acetone. The pellets were further centrifuged' and washed several times with ice-cold acetone. Finally, the protein was dissolved in 350 μl of a rehydration solution containing 8 M urea, 2% CHAPS, pH 4-7, 5% ampholyte, and 20 mM DTT, and then oscillated and placed at room temperature for one night. The rehydrated solution was centrifuged at 12,000 rpm for 20 minutes' and the protein concentration in the supernatant was adjusted to about 500 y g/350/zl.

Isoelectric focusing (IEF) was performed on an IPGphor electrophoresis unit (Amersham Biosciences) using a ready-made Immobiline Dry-Strups strip (Amersham Biosciences) having a pH gradient of 4-7 and a length of 18 cm. The strip was rehydrated by application of a constant low voltage (30 V) for 20 hours in the presence of the rehydration solution. The IVI of the first dimension was carried out at 18 ° C by a preset voltage of 0.5 hours 500 V, 0.5 hour 1000 V, 0.5 hour 1500 V, 0.5 hour 2000 V, 0.5 hour 3000 V, 1 hour 6000 V, and 10 hour 8000 V. After the end of the first dimension of IEF, the strip is contained in 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% Wv glycerol, 2% w/v SDS, and dithioerythritol (DTE). Incubate for 15 minutes in equilibration buffer. Immediately thereafter, it was cultured in an equilibrated buffer of iodoacetamide for 15 minutes. Then use Protean II xi Multi-Cells (Bio-Rad Laboratories, Hercules, CA, US A) to carry out the second-dimensional electrophoresis at a current of 22 1345472 10 mA at a concentration of 22-185% at a concentration of 8-18%. When a current of 35 mA was applied until the uranium margin of the buffer was 5-1 〇 111111 from the bottom of the gel, protein staining was performed with Sypr〇Ruby. The electrophoresis pattern of the 2_D gel was compared and analyzed using PDQUEST software version 6.2.1 (Bio-Rad Laboratories). To compare the protein in the -S gel, the total intensity of all protein spots appearing in the gel was normalized. The relative intensity of the protein spots was then statistically analyzed by the nonparametric test of Mann and Whitney. A P value less than 0.5 is considered to be statistically significant. For mass spectrometry, the locked protein spot is removed from the gel and then washed with 50 mM ammonium bicarbonate (amm〇nium bicarbonate) before the gel digestion. % acetonitrile (acet〇nitrile) is cleaned. After washing, the gel pieces were treated with acetonitrile, dried, and rehydrated with 50 mM hydrogen carbonate containing 〇 01% of sequence grade trypsin (Promega, Madison, WI). After hydrolysis of the enzyme at room temperature overnight, the peptide was extracted twice with 50% acetonitrile and 0.1% trifluoroacetic acid, followed by shaking and ultrasonic treatment (20 minutes each). After drying, the peptide mixture was suspended in 10# 1 of 50% acetonitrile and 5% TFA, and subjected to ultrasonic treatment for 30 seconds for direct spotting, and subjected to MALDI and API-TOF analysis. Mass spectrometry can be performed at the core laboratory of the Center for Protein and Structural Biosciences (Academia Sinica, Taipei, Taiwan). • Western dot ink method: all Western ink and ink laws in this invention are based on

Hsieh et. al. (2000) Ga Ge (10) 24: 66-70 description. Membrane with direct anti-SMN (1:10,000, BD Transduction Laboratories), anti-cytoplasmic protein (1:10,000, Calbiochem), anti-a-tubulin (1:30,000, Amersham Pharmacia Biotech), anti->5- Actin 23 1345472 (1:100,000, Sigma), Calnexin (1:500, Chemicon) and Cleavage-caspase 3 (1:1000, Cell Signaling) The antibodies are cultured together. The Western blotting method is performed by a computerized densitometer via a scanning autoradiograph. The signal intensity was measured by a Phoreticx 1D program (Phoreticx International) by density measurement analysis (FujiFilm LAS-1000 plus pictography).

Immunocytochemistry: NSC34 cells or primary cell neuron lines grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. ), then blocked with 3% BSA in PBS. Add anti-SMN, anti-ChAT (Chemicon), anti-GOLPH (Shang's body label, Abeam), anti-Neuroflally H (Chemicon), anti-tubulin, anti-ySIII-tubulin (Chemicon), anti-flag (Sigma), and a primary antibody against cytosolic protein (Calbiochem) (diluted to 1:500), followed by the addition of a suitable secondary antibody (Molecular Probe, Invitrogen) conjugated to the fluorescent stain. Finally, samples were processed via DAPI (Sigma) for 5 minutes to identify nuclei and mounted with a fluorescence mounting solution (DAKO). Confocal images were obtained via an LSM510 Meta Confocal Laser Scanning Microscope (Zeiss, Oberkochen, Germany). Then use the LSM5 Image Browser software for image acquisition. Immunohistochemistry (IHC): A mouse model of spinal muscular atrophy that can be used for research according to Hsieh-li et. al. (2000) 24: 66-70 and Li et al. in June 2001. U.S. Patent No. 6,245,963, issued toK. The thickness of the spinal cord and cold bead slice is anti-cytosine (1:400 ' Calbiochem), anti-ChAT (1:200, Chemicon), and anti-de- wintering enzyme 2 (caspase 2)

The primary antibody (1:200, Chemicon) was cultured and then cultured with an appropriate secondary antibody (Molecular Probe, Invitrogen) in combination with a fluorescent dye. Finally, to identify the nuclei, the samples were incubated with DAPI (Sigma) for 5 minutes and then mounted with fluorescent mounting (DAKO). The negative control group was cultured by substituting the aforementioned antibody with an IgG antibody of the same species as the primary antibody. Conjugate focal images were obtained via a LSM510 Meta co-focus laser scanning microscope (Zeiss, Oberkochen, Germany). Then use the LSM5 Image Browser software for image acquisition. Whole silk fibroin (α: Umbrella) - wh〇le-mount staining: for immunostaining of whole specimens, dissected and removed at embryonic stage 18.5 (embryonic stage, E18.5 The spinal muscular atrophy mice and their control group were the diaphragm muscles of the mouse embryo. The tail of each embryo was cut and subjected to genotyping analysis as described by Hsieh et. al. (2000) Ge «W24: 66-70. Immunostaining of the diaphragm muscle was performed as described by Lin et. al. (2005) Wwr (10) 46: 569-79. A total of confocal micrographies were obtained from a Zeiss LSM5 PASCAL microscope. Subcellular fractionation of spinal cord cell lysate: spinal cord tissue obtained from mice with type 1 or type 2 spinal muscular atrophy mice and their control littermates. Kikuchi et. al. (2006) i〇3: 6025-30 performs subcellular separation to form the Cyt〇slic fraction and the microsomal fraction 25 1345472 (microsomal fraction). Take the same amount of protein for Western blotting analysis. result

Production of NSC34 cells that can induce SMN knockdown by deoxy-hydroxytetracycline: NSC34 cell line fuses neuroblastoma cells with mouse primary motor neurons, which have some similar motor neuron characteristics. Including the cholline acetyl transferase and the expression of neurofilament triplet protein as described by Cashman et. al. (1992) Dev 194: 209-21. The knockout cells can be used as a useful tool in studying the function of motor neurons. The manufacture of SMN-declining NSC34 cells accurately mimics the symptoms of SMN deficiency. In these cell lines, proteins that may be involved in the pathogenesis of spinal muscular atrophy and which are regulated differently can be identified by proteomic analysis. The SMN shRNA vector was designed to knock down the SMN gene expression in NSC34 cells. The NSC34 cell line was transiented with the existing SMN shRNA vector and all proteins were extracted, and the SMN expression was estimated by Western blot analysis. After 48 hours, the amount of SMN protein in the transfected NSC34 cells decreased with SMN shRNA compared to untransfected NSC34 cells (Fig. 1 (A)). This result was confirmed by immunocytochemical analysis, which showed less SMN in the cytoplasm or intranuclear gem dot structure after transfection with SMN shRNA compared to the cells that were not transfected. Performance (Fig. (B), circled in white). θ Recent studies have shown that cells that are completely deficient in SMN will undergo apoptotic cdl death, with reference to the splicing of the squadrons (fine 3) 26 1345472, the descending plants after treatment with deoxy hydroxytetracycline, and Compared with the cells treated with deoxy-hydroxytetracycline, the cytoplasmic prion protein expression was significantly increased. In the class, the cytoplasmic sputum protein 1 in the spinal cord of mice with myeloid muscular atrophy was analyzed by Western blotting. The spinal cord extracts of four first-type hippocampus muscle atrophic mice and their control littermates were isolated: and the anti-cell 燐 protein antibody was used for immuno-point ink analysis. As a result, the expression of cytosolic prion protein in the first type of spinal muscular atrophy mice was also increased (Fig. 2(C)). To confirm whether cytoplasmic prion protein is only expressed in the spinal cord of mice with spinal-like muscular dystrophy, the analysis of the expression of cytoplasmic prion protein in the cerebellum H, brain extract and anti-antigen The antibody was subjected to immuno-point ink analysis, and the results showed that the amount of cytoplasmic prion protein in the mice of the second-concretion mice and the control group was in the affected part __ cells (four) "amount of 2 襞燐 protein The upward ascending line is associated with spinal muscular atrophy. The cytoplasmic prion protein is accumulated in the NSC34 cells of the spine-like muscle dystrophy (4). In order to judge the position of the cytoplasmic protein in the spinal cord, the position will be increased. - The type of spinal muscular atrophy mice and their control group were treated with immunohistochemical staining with antibodies against acetylcholine transferase (type of motor neuron) and cytoplasmic prion protein. The accumulation of prion protein occurs in motor neurons of mice with spinal age-deficient muscle wasting (figure 3, indicated by arrows) but will not appear in the control group (third image, read head). S ) 28 U4M72 = The average number of motor neurons in the grade and spinal-like muscular atrophy mice was 835.5 and 617.5. The cumulative percentage of cystein in the motor neurons of the control mice was 17.7%. The increase in type I spinal muscular atrophy in mice was 12.3% (Fig. 3 (B)). The cytoplasmic protein accumulation in mice with spinal muscular atrophy was not measured by quantitative analysis. The amount is more than six times that of the control mice (Fig. 3 (B))

The median Μ5±5.6% was 74.25±15.7% in the first type of spinal muscular atrophy mice. After treatment with deoxy-hydroxytetracycline to reduce SMN performance, NSC34 cells were harvested for immunocytochemical analysis. The cytosolic protein intensity of both the control and SMN knockdown NSC34 cells (Fig. 3(c)) was measured by MetaMorph software. Compared with the control cells, the cytoplasmic prion protein signal of the 8 ΜΝ decimated cells increased (Fig. 3). This data shows that the SMN phenotype is associated with an upward increase in cytoplasmic prion protein. .

Excessive expression of cytoplasmic proteins disrupts microtubule tissue and axon growth. Factors that disrupt microtubule stabilization (such as cytosolic prion protein) can cause microtubule tissue disorders and depolymerization of nerve cells, leading to neuronal degeneration. To test whether the up-regulation of cytosolic protein levels caused microtubule de-assembly, the cytosolic protein carrier was introduced into NSC34 cells. The microtubule morphology of NSC34 cells was analyzed by immunocytochemistry. As shown in the fourth panel (A) (and the white circle in the left panel, the white circle shows an enlarged view), the excessive expression of cytosolic prion protein in NSC34 cells reveals a destructive tissue pattern of microtubules. In normal cells transfected with cytosolic prion protein, the microtubule structure is normal (indicated by arrows). F-actin staining showed that NSC34 cells transfected with cytoplasmic prion protein showed abnormal (S) 29 1345472 neurite branching compared to untransfected cells (Fig. 4 (A) (B)). This data shows that Cong Meimei plays an important role', that is, the organizer of microtubules. Baiping, NSC34 cells with anti-α-tubulin analysis of SMN knockdown showed a violation of the two groups. In contrast, some cells appeared from the cytoplasm to the fine 'continuous nerve filaments (Fig. 4C) White circle). This data = NPV34 cells with excessive expression of prion protein showed an abnormal situation in microtubules, and the amount of cytoplasmic prion protein increased upwards to motor neurons = &

The diaphragm muscle of mice with spinal muscular atrophy shows axonal disease, and the destruction of cytoplasmic protein in the microtubule structure is detrimental to the length of the motor neuron sleeve. This led to a study of axonal extension defects in spinal cord muscle-deficient mice. The performance of cytoplasmic prion protein in the spinal cord at different embryonic stages was determined. At the embryonic stage of 12·5 (Ε12.5), 15.5 (Ε15.5), and 19.5 (Ε19.5), spinal cord extracts were obtained from spinal cord muscular atrophy and its control littermates. The cytoplasmic prion antibody was used for immunoblotting. The results showed that there was no significant difference in the expression of cytosolic protein at 12.5 ( (1.35 〇 13% in the control littermates, and ι in the spinal dystrophic embryos). Μ0.1%). However, compared with the control group litter + mice, mice with spinal muscular atrophy were found to have higher cytosolic protein expression at Ε15.5 and Ε19.5, which was at Ε15.5. In the same group of mice, I.13 soil was 0.06°/〇, 1.4±0.11% in embryos with spinal muscular atrophy, and 0.95± in the control group. 0.05%, L2±0.03〇/〇 in embryos of spinal-like muscular atrophy (fifth (A). The whole specimen of the diaphragm muscle was stained with anti-neurofilament antibody, and the control group was analyzed. And the first type of spinal muscular atrophy mice

30: The presence of the f protein around the nucleus is consistent with the distribution of high-kilten bodies (Strey et. al. (2004) Orthopaedics / 165: 1701-18) 〇 $ scale, white line in the spinal cord The cytoplasm of mice with muscular dystrophy is cumbersome: and these accumulated cytoplasmic proteins may be associated with high kiln. The cytosolic protein was analyzed by Western blotting on the spinal cord lysate of the mice of the genus Zhao, = meat-deficient mice and their control group. This result shows that in the first type of spinal muscular atrophy mice, the expression of the eight-pulp protein is not only increased in the cytoplasmic fraction but also in the microsomes (Fig. 7(A)). Segmented high-kilten bodies are present in cells with excessive expression of cytosolic prion protein (seventh panel (B)). The antibodies of the first type of spinal cord muscle, the mouse, and the control group of the spinal cord of the littermate of the mice were immunologically colored. The triple labeling of ChAT, and the protein and caspase 2 showed that most of the cells with caspase 2 =% are motor neurons with cytoplasmic protein accumulation φ χ 槿 导, field cells The music of apoptosis (aP〇Ptosls) was analyzed by Xigu ν54. The drugs that are administered to induce these cells are encapsulated in the spear, and the amount of protein is expressed and money (4). This data = the accumulation of =: causes the implementation of the ridge and muscle atrophy and the examples are for illustrative purposes and are not intended for use in the scope of the present compositions and methods. Some implementations = the same method (4), modified, or (4) can be implemented in the middle, and obtain substantially similar results. 1345472 Sequence Listing <110> Central Research Institute <120> Diagnosis of spinal muscular atrophy and its treatment <140> TW 96147825 <141> 2007-12-14 <150> US 11/703,809 <;151> 2007-02-07 <160> 6 <170> Patentln version 3.3 <210> 1 <211> 6762 <212> DMA <213> Homo sapiens <220><221> Feature <222> (1).7(6762) <223> genomic DNA of cytosolic protein STMN1 gene No. NC 000001.9. .gi:89161185)

Genbank >>>> 0 12 3 2 2 2 2 2 2 2 2

Misc-feature(2202). (2204) Start codon atg2202-2204 0 12 3 2 2 2 2 2 2 2 2 <<<<

Misc-feature (2202).. (5862) The coding sequence of STMN1 is a combination of nt22〇2-22l4, nt5791. .5862. 55-3237, nt5196-5387, and <220><221><222><223>

Misc-feature (5860) .. (5862) stop codon taa5860-5862 < 400 > 1 aggggcactg ctctgtccga gtgctgccct tggggcgag gtggagtcca ggcggccaaa gtttggaaag gtagggaag ctccgccctg cccttgttct cgagaatggg gagctggtt tgccacgccc tcttccacgg cgagaccacc cccctagtc ctccccaggc gccctgcaac ccccggcatt gtcctcctg ggccc cgggcatgtg gctctacaag 60 acccccccgc cctccgcctg 120 ggacctagtc Cggggtccac 180 caggcccaca cctggggatg 240 cctccgggac aggactacac 300 360 360

1345472 ttcccgaggt gcttcggggt cccggggggc ggcgctccac gcgggttgtg gggggcgggg gcggcacgtg ccgccgctct cggccaatgc ggagccccgc gcggaggtca cgtgcctctg tttggcgctt ttgtgcgcgc ccgggtctgt tggtgctcag agtgtggtca ggcggctcgg actgagcagg tgggtgcggg gctcggagga ggcggcggct ggctgaggcc agcaagaggg acgcggtcgg cgggaggggc tgggccgtgg cagcgacccc ctgctgcagg gcggcgggcg gggctgcggg cctcggaggg gttggtgggc gggggtcgct ccgctttgtg tgtggctcgg gcggagcctc gcctttgtcc ccgctctccg ggggcgcggc tgttcgtggg cagggggctg ggcgatcacc gggcgtccgc tccggggtgc cgtcgaggag acaatagggg gcgtgggccc tcgtttacct ccctccctcc ctcccttccc tgcgggcccc gccgggttcc ccattgtctg aagggacggg gcggtgcccc agggaccagc ggctttagga ccaaactgcg ggcagccagg gccgcgaccc tccctgcgac cgtcccctgg cgaccgcagc tggtgattga ggggcggcgc tcccgggccc cacgagggtt cttctgtctt cgcggccgga cgcgcggaca gcgtgggtgg cggcaggttg ggcatgggga cggcgggagg cggtggcgag ctcaccgcgg gaccacccgg gggcctgttc ccggggcctg ccccacccgc tgaactgtga agggggtggt ggcggcggcc tggaggtgtt tttggcggga gttggggggg gcgtccgcgc agggggagtc aggcaggggc gg agttaccc ggattggacc gttagccccg cccacccctc cccttcccac gcgcgcgggc tccggggtgt tgagttcggg gagattcgaa aaggcgcggg gaggaagggg gcggggccag gggccggagc gcaaggcgtg ctctgattgg ccgggggcga ccggtcctct tttcctcgcc cggaccaggg.ccacgcccat cctgggtccg gtgctgcgtc taattctctg cttttcttaa atcttgtcgc tgcctctgat tttaattcct agcttttggg aacctgtcat cctacgtttt tggtactagc tggcgtctac aaaagtcata atgttaaaaa gatcaacaag agatacagca tttttcatga cataacggca gcaaatataa gtcaaaatct agaggttcat aaacattttg cttgctgttg ggcaaggaag cttaaacctg agggacaata ggagttcaac attattggtt actattagct tgggcgtttt cttatccacc acgtcagaca cagacaaagc aggggtgggt atttcatttg cacaatgagt tgtaggcagt attaagatgg ctccgggggc actgttgagt tgaatctgga atatcttctt acagtttcgg tgaaatgtta aagagtttat gggggaaaaa ttcttcaccc ttgtgacttt gtctgatttt aaaaatccaa gagttttatg accgagaaag ctcagttaac ttgattttct ggaaccaata tcatattcag gtcatatttc ccaatgttta 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 I860 1920 1980 (:S ) 2 1345472

tttagtagat ttctgacgat caaagaaagt ttgtgcagaa aatctatttg gtcttcgcct gagaagaatt acagtttggc gcgattgtca gtttttctgg tggagtgcag tcaagcgatt acccacctaa tggtctcaaa tataggcatg ctgcaaatac cacaactttc atagtacagt ctagatatcc ctcagccctc gatctttccc aacgaagcaa tctataacag tggaaggaaa tatttttctc gaatgttaag ccctggtcta gttgccagat taaattgcta tttgataatt gtatgtagat gattgcatgt tacactgcct gaaaatctga ttgatttggc taaaaatgat ttagtctgaa gatactgatg gagatggttt tggcttaatt ctcccacctc tcaaaaaaaa cttcagggct agccatggta atcttcccct ctcatcagcc ttattagaac aggtgaaaga ggtcaaaaga tggaggaaat ttcacagaaa gaagaaaaca taccagtcct ctttaataca tcgtttttct tcttaaacca gattctttgg gtgtgattgt tttttcgtgg gtgatgtgag ttttgaaaat gtcgcttgtc aattgtaatg acttatgttt gcaaaattgt tctcaaaaca ttattttttt atagctcatt agcctccata ttttttttgt caagggatct cctggccttg agtgtcagta ctagtttaat aggagaatcc actggagaag atctgttcca tcagaagaaa gcaggatatt gaattgtagc catttattga tggtgttgcc gcagtcatac aagctcatgc cactttggtt gttgctcagc ttaatttaga atttttttgg cattttcagg ttctattcac tatcacttga ggcttatgat tggtcttacc atgtgccgag ggagtttgca gtatttatta tgagacaggg gcagccttga gcatctggga agagatgggc tcccatgttg ggaaatggta gactgatagg acattaaatt tggttttctg cgtgcctcag gaattccccc ttagaagctg aatttatgta tacactgtga actcctgtta tcaaataatg aactagtaag ttctctcatg ttgttttttg tggtatcatg cgtctttatt gttgatgaca actttcctta catggcttct tttagattga aaaacctatt agctcgtaat caagtctttt atccaaaaat tctcaccttg cttctggagc ctatcgacat tttccctttg gcctcccacg tttagataat aataaaaagg gatttgggtt aattataaat gccaggcttt tttcccctcc cagaagaaag atgggcagat ttattacata catgcccgct gaaattagaa tgttggggtc cttcctcttt agagctgtac gtagcttttc ccacgtattt tatagaaagg tcccagttga tctggtaggt gatggctcag ttatgaatag tgtttattgg gtttatgaaa gttgagcttt ttgcccaggc tcaagtgggc gggccaccac ttgcccaggc gtgctgggat aatatcttgc ggaaaaaaaa ttagaaaatt ataatcaatt tgagctgatt aaagaagaag acgcaaggta caattttatt tgccagtgac cctttgttta acagtttcag agaattcaag gaaaagattt aataacattt tcttatctaa 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3 060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 (S > 3 1345472

acaattctat tatgctagtt cattatcccg atgttactca ataggattga cagaccagca cactaaggag accaaaggtt ctgtgcttta cagaagagaa tccaatgtgt taattaatgc tgtcttaaat tggttaaaat aattctttac ttaagcagta ataagcactt tttatgatag gccccttttc attttatttt gttcactgca acttagacta ggatttcacc ttggcctccc ttagagtgcc agctggctga acaacttcag accgagaggc tataaagtaa gtagaaaatt cttccgagaa ttaacaactg ttttaaagct tagtgatttg tcgtcttcta ggacagtgga catagtaact tcaatagaac tttgagaaca agtttcctga agtaatctac ttttacaatg aataaaagtg aatcttacaa ttccaaagtt ggataattca aatcccctta ttgaggcaga acctccacct caggcacccg atgttggcca aaagtgctgg tagcctatta gaaacgagag taaaatggca acaaatggct ctatctttaa tgggaaatac tgcaatagtg gcaaccagtg tacataactt ggggcaagtg tagtatacac ttaacatttt ttcatttaac aggcacaaaa aagcttttta ggctaaatat cagtgtcaga agctgttctg gacaaaagct agacttagtc agctagtttt ccactcgcaa ggatttcctt aaatacaaat ggtcctaatt gtaaattatt gtctctgtca cccgggttca ccaccacgcc ggctggtctt gattacaggc aacttttttt cacgagaaag gaagagaaac gccaaactgg aagctaatca agaaatttat acacacccgc tgagttcatg gacatcaacc aatgaaactc tgctctgtgg ttgaaggggt aataagcctt ttgaatttcc ctttgggtag gtacatattc aaggactaat actagaaaaa ttggtgttat tttattgtaa cccagggtgg agcaattctt cggctaattt gaactcctga gtgagccacc tccagtccca aagtgcttca tgacccacaa aacgtttgcg gaagaatcaa aaatggaaat ggtgtgtttc tccctttctc ggtaaggtaa ccgaagagcc gagtaggagg ctggccctgt ccatcatgac ttatcctgca cataccaagg ttccttgaaa tatcattgtt cttttgaagt ctcaagtcat tgggtaagat gaatatcatt ttaaagggac ttttaactta agtgcagtgg ctgcctcagc ttgtattttt cctcaaatga acacccagcc tgaagctgag gaaggcaata aatggaagct agagaaggtt taaatattaa taaaagtatt tttccattca aacaaccctg taaccttccc tcagtgctta aagcaataca agtcattagc taggatttgg agtcagtggt tatggtctgg caaatttttc ttatcagtta taaatagtga ccagatacca ttctgaacag ttcacagtat cttattttct tgctgaatac cacgatctca ctcccaagta agtataggta tccacccacc tgaatacatt gtcttgaagc gaagagaaca aataaagaga ggtttcttac 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 4 1345472 tt tgtaaaag ggttgagctt ggagtttgat gcaccaatga gttggcttga actaagtgct 5460 ttgataaaag gtgtttggtg tctttttgtc atccattttg gggcttaaca tattaaatga 5520 aaggtatatt ttaagatgga atattcagta attcccagca taattgcaca gtccttggaa 5580 gtccagtagg cagcttgtta ggttctacaa gggacccagg agatttgatg atgatgtctc 5640 agaacttaaa ttgtgtggtt cccacaggct gtaatatatg cactgaggtt gtgttgggcc 5700 tctttgaggt gggggctggg ggtcgtgact tgacaggctt tttttttttt tttttttttt 5760 gactgatgac accttaccct tcctttacag gataagcaca ttgaagaagt gcggaagaac 5820 aaagaatcca aagaccctgc tgacgagact gaagctgact aatttgttct gagaactgac 5880 tttctcccca tccccttcct aaatatccaa agactgtact ggccagtgtc attttatttt 5940 ttccctcctg acaaatattt tagaagctaa tgtaggactg tataggtaga tccagatcca 6000 gactgtaaga tgttgtttta ggggctaaag gggagaaact gaaagtgttt tactcttttt 6060 ctaaagtgtt ggtctttcta atgtagctat ttttcttgtt gcatcttttc tacttcagta 6120 cacttggtgt actgggttaa tggctagtac tgtattggct ctgtgaaaac atatttgtga 6180 aaagagtatg tagtggcttc ttttgaactg ttagatgctg aatatctgtt cacttttcaa 6240 tcccaattct gtcccaatct taccagatgc tactggactt gaatggttaa taaaactgca 6300 cagtgctgtt ggtggcagtg acttcttttg agttaggtta ataaatcaag ccatagagcc 6360 cctcctggtt gatacttgtt ccagatgggg cctttggggc tggtagaaat acccaacgca 6420 caaatgaccg cacgttctct gccccgtttc ttgccccagt gtggtttgca ttgtctcctt 6480 ccacaatgac tgctttgttt ggatgcctca gcccaggtca gctgttactt tctttcagat 6540 gtttatttgc aaacaaccat tttttgttct gtgtcccttt taaaaggcag attaaaagca 6600 caagcgtgtt tctagagaac agttgagaga gaatctcaag attctacttg gtggtttgct 6660 Tgctctacgt tacaggtggg gcatgtcctc atcctttcct gccataaaag ctatgacacg 6720 agaatcagaa tattaataaa actttatgta ctgctgtagc aa 6762 0 12 3 1 1 1 2 2 2 2 2 V << 2 1730

DNA

Homo sapiens <220><221> Misc_feature <222> (1).7(1730)

S 5 1345472 9 <223>STMN1 mRNA of Peng^ pity protein (the longest change in Genbank No. NM_203401.1-.gi:44890051 - allogeneic, with reference to variants 2 and 3. ' <220> <;221> Misc-feature <222> (3657..(814) <223> coding sequence of the cell line protein STMN1 protein <220>

<221> CDS <222> (365)..(814) <400> 2 60 120 180 240 300 360 409 457 505 553 601 649 atcaccgggc gtccgctccg gggtgccgtc gaggagacaa tagggggcgt gggccctcgt

ttacctccct ccctccctcc cttccctgcg ggccccgccg ggttccccat tgtctgaagg gacggggcgg tgccccaggg accagcggct ttaggaccaa actgcgggca gccagggccg cgaccctccc tgcgaccgtc ccctggcgac cgcagctggt gattgagggg cggcgctccc gggccccacg agggttcttc tgtcttcgcg gccggacgcg cggacagcgt gggtggcggc aggactttcc ttatcccagt tgattgtgca gaatacactg cctgtcgctt gtcttctatt cacc atg get tet tet gat ate cag gtg aaa gaa ctg gag aag cgt gee Met Ala Ser Ser Asp lie Gin Val Lys Glu Leu Glu Lys Arg Ala 15 10 15 tea ggc cag get ttt gag ctg att etc age cct egg tea aaa gaa tet Ser Gly Gin Ala Phe Glu Leu lie Leu Ser Pro Arg Ser Lys Glu Ser 20 25 30 gtt Cca gaa ttc ccc ett tee cct cca aag aag aag gat ett tee ctg

Val Pro Glu Phe Pro Leu Ser Pro Pro Lys Lys Lys Asp Leu Ser Leu 35 40 45 gag gaa att cag aag aaa tta gaa get gca gaa gaa aga ege aag tee

Glu Glu lie Gin Lys Lys Leu Glu Ala Ala Glu Glu Arg Arg Lys Ser 50 55 60 cat gaa get gag gtc ttg aag cag ctg get gag aaa ega gag cac gag His Glu Ala Glu Val Leu Lys Gin Leu Ala Glu Lys Arg Glu His Glu 65 70 75 aaa gaa gtg ett cag aag gca ata gaa gag aac aac aac ttc agt aaa Lys Glu Val Leu Gin Lys Ala lie Glu Glu Asn Asn Asn Phe Ser Lys 80 85 90 95 atg gca gaa gag aaa ctg acc cac aaa atg Gaa get aat aaa gag aac 697

Met Ala Glu Glu Lys Leu Thr His Lys Met Glu Ala Asn Lys Glu Asn 100 105 110 ega gag gca caa atg get gee aaa ctg gaa cgt ttg ega gag aag gat 745

Arg Glu Ala Gin Met Ala Ala Lys Leu Glu Arg Leu Arg Glu Lys Asp (s > 6 793 793

1345472 115 120 125 aag cac att gaa gaa gtg egg aag aac aaa gaa tee aaa gac cct get Lys His lie Glu Glu Val Arg Lys Asn Lys Glu Ser Lys Asp Pro Ala 130 135 140 gac gag act gaa get gac taa tttgttctga gaactgactt tctccccatc Asp Glu Thr Glu Ala Asp 145 cccttcctaa atatccaaag actgtactgg ccagtgtcat tttatttttt ccctcctgac aaatatttta gaagctaatg taggactgta taggtagatc cagatccaga ctgtaagatg ttgttttagg ggctaaaggg gagaaactga aagtgtttta ctctttttct aaagtgttgg tctttctaat gtagctattt ttcttgttgc atcttttcta cttcagtaca cttggtgtac tgggttaatg gctagtactg tattggetet gtgaaaacat atttgtgaaa agagtatgta gtggcttctt ttgaactgtt agatgctgaa tatctgttca cttttcaatc ccaattctgt cccaatctta ccagatgcta ctggacttga atggttaata aaactgcaca gtgctgttgg tggcagtgac ttcttttgag Ttagtttaat aaatcaagcc atagagcccc tcctggttga tacttgttcc agatggggcc tttggggctg gtagaaatac ccaacgcaca aatgaccgca cgttctctgc cccgtttctt gccccagtgt ggtttgcatt gtctccttcc acaatgactg ctttgtttgg atgcctcagc ccaggtcagc tgttactttc tttcagatgt ttatttgcaa acaaccattt tttgttctgt gtc Cctttta aaaggeagat taaaagcaca agcgtgtttc tagagaacag ttgagagaga atctcaagat tctacttggt ggtttgcttg ctctacgtta caggtggggc atgtcctcat cctttcctgc cataaaagct atgacacgag aatcagaata ttaataaaac tttatgtact gctgtagcaa aaaaaaaaaa aaaaaa <210> 3 <211> 149

<212> PRT <213> Homo sapiens <400> 3

Met Ala Ser Ser Asp lie Gin Val Lys Glu Leu Glu Lys Arg Ala Ser 15 10 15

Leu lie Leu Ser Pro Arg Ser Lys Glu Ser Val 25 30

Pro Glu Phe Pro Leu Ser Pro Pro Lys Lys Lys Asp Leu Ser Leu Glu 844 904 964 1024 1084 1144 1204 1264 1324 1384 1444 1504 1564 1624 1684 1730

Gly Gin Ala Phe Glu 20 (S ) 7 1345472 35 40 45

Glu lie Gin Lys Lys Leu Glu Ala Ala Glu Glu Arg Arg Lys Ser His 50 55 60

Glu Ala Glu Val Leu Lys Gin Leu Ala Glu Lys Arg Glu His Glu Lys 65 70 75 80

Glu Val Leu Gin Lys Ala lie Glu Glu Asn Asn Asn Phe Ser Lys Met 85 90 95

Ala Glu Glu Lys Leu Thr His Lys Met Glu Ala Asn Lys Glu Asn Arg 100 105 110

Glu Ala Gin Met Ala Ala Lys Leu Glu Arg Leu Arg Glu Lys Asp Lys 115 120 125

His lie Glu Glu Val Arg Lys Asn Lys Glu Ser Lys Asp Pro Ala Asp 130 135 140

Glu Thr Glu Ala Asp 145 <210> 4 <211> 59 <212> DNA <213>Artificial Sequence<220><223:> Chemically synthesized oligocytic acid <400> 59 gatcccgtag ctaatagtac agaacttcaa gagagttctg tactattagc tactttttt <210> 5 <211> 58 <212> DNA <213>Artificial sequence <220><223> Chemically synthesized oligonucleotide <400> Agcttaaaaa agtagctaat agtacagaac tctcttgaag ttctgtacta ttagccat 58 <210> 6 8 1345472

<211> 16 <212> DNA <213> Artificial sequence <220><223> Chemically synthesized oligonucleotide <400> 6 ttgcgagaga aggata 16 (S ) 9

Claims (1)

Patent application scope of May 3, 100, Republic of China: 1. A screening method for diagnosing spinal muscular muscular atrophy (SMA) or low SMN protein-based condition, which includes the following Steps: (A) providing an anti-cytoplasmic protein antibody and a sample of spinal cord cells obtained from a human patient, wherein the aforementioned human patient is at least suspected of having spinal muscular atrophy or a condition caused by low motor neuron survival protein, (B) contacting the aforementioned anti-cytoplasmic protein antibody with the aforementioned spinal cord cell sample under conditions in which the binding agent can specifically bind to the cytoplasmic protein in the sample, and the complex is produced without removal. Combining the binding agent to leave the remaining complex; and (C) detecting the remaining complex, and comparing the detected amount of the complex or the corresponding cytosolic protein concentration in the spinal cord cell sample obtained from the human patient with the healthy spinal cord a control amount in the cell sample, wherein the detection amount of the aforementioned complex or the cytosolic serotonin concentration is higher than the control amount in the aforementioned healthy spinal cord cell sample As a positive screening result. 2. The method of claim 1, wherein the anti-cytoplasmic protein anti-system is an anti-cytoplasmic antibody having a detectable label attached thereto. 3. The method described in claim 1, wherein the screening of 1345472, the results of the May 3, 100, and the combination of further test results to diagnose spinal muscular atrophy or low motor neuron survival protein The resulting condition; and the foregoing further test results include functional motor-meta-viable protein expression, intranuclear "gem" dot-like structure (nuclear gem), snRNP formation, or a combination thereof. 4. A kit for screening for conditions caused by spinal muscular atrophy and low motor neuron survival, comprising an anti-cytoplasmic antibody and instructions for its use. 9. The kit of claim 4, wherein the anti-cytoplasmic protein anti-system is an anti-cytoplasmic antibody having a detectable label attached thereto. 6. The kit of claim 4, wherein the foregoing instructions further comprise a combination of the screening results and further test results to diagnose spinal muscular atrophy or low motor neuron survival protein. The resulting condition; and the foregoing further test results include functional motoneuron survivin expression, nuclear "gem" dot structure appearance, snRNP formation, ® or a combination thereof. 2 1345472 96 (Hr 6 white (C) ϊ| 1 :.,〇r ___ ..-, *- v T: brother.:j; __ sealing.:;; ___ 7.丄:',) Ben 4-,仁1 1 '> 9 :·!;] 1 I · ^1·· —mu ...... I 'TLi-v ;tiT ii (D) Jj towel along '-1' Table 1 1 1,. i,:,1 ! : Y 夂^ is .-· LV ::;; ___ .: η 一:::ί 一 £我3 V 呔$ Mumu 1, :!ru second picture